TGFBI R124H mutant allele silencing in granular corneal dystrophy type 2 using topical siRNA delivery

Andrew McLain; Amanda Kowalczyk; Paulina Baran-Rachwalska; Flavia Maria Sutera; Louise J. Robertson; Nadia Sukusu Nielsen; Jan J. Enghild; Diego Cobice; Filippo Bonelli; Vanessa Barbaro; Stefano Ferrari; Benjamin Patterson; Luca Moore; John Marshall; M. Andrew Nesbit; Tara Moore

doi:10.1016/j.jconrel.2025.113681

TGFBI R124H mutant allele silencing in granular corneal dystrophy type 2 using topical siRNA delivery

Andrew McLain, Amanda Kowalczyk, Paulina Baran-Rachwalska, Flavia Maria Sutera, Louise J. Robertson, Nadia Sukusu Nielsen, Jan J. Enghild, Diego Cobice, Filippo Bonelli, Vanessa Barbaro, Stefano Ferrari, Benjamin Patterson, Luca Moore, John Marshall, M. Andrew Nesbit, Tara Moore^*

^*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

Abstract

In recent years, success has been achieved in treating several eye conditions with oligonucleotide-based therapies. Herein, we outline the experimentation involved in progressing selection and development of a lead therapeutic siRNA for R124H mutation of TGFBI gene which causes Granular Corneal Dystrophy Type 2 (GCD2/Avellino CD). Firstly, a series of siRNA designs, generated by a gene walk across the R124H TGFBI mutation site, were tested and a lead siRNA identified. The lead siRNA was delivered into an immortalised human corneal epithelial cell line to assess on-target efficacy and off-target effects. The in vivo efficacy of the lead R124H TGFBI siRNA, complexed with Bio-Courier technology, silicon stabilized hybrid lipid nanoparticles (sshLNP), was assessed in a mouse model of GCD2 which expressed the human R124H TGFBI transgene. Following topical siRNA application for 5 consecutive days, expression of the R124H mutant TGFBI transgene was measured and shown to be reduced by 22.4 % (± 15.7 %, p < 0.05). We investigated gene expression in the mouse cornea and showed expression of murine Tgfbi was 20-fold lower than TGFBI in human cornea, and expression of the mutant TGFBI transgene was a further 3-fold lower. This estimated 60-fold lower mutant transgene expression may explain the low frequency of corneal deposits observed in this mouse model, limiting its usefulness to test whether siRNA silencing is capable of phenotypic improvement or regression of GCD2/Avellino corneal dystrophy. We assessed WT TGFBI silencing in human primary corneal epithelial cells (PCEC) derived from human corneal limbal biopsy material, which express TGFBI at a similar level to human corneal biopsy. We demonstrated that a single 100 nM siRNA treatment, delivered by the sshLNP to the primary human corneal epithelial cells, gave 26.6 % (± 6.6 %, p < 0.001) reduction in TGFBI mRNA and a 15.4 % (±10.5 %, p < 0.05 %) reduction in TGFBi protein after 48 h. In consideration of the mutant gene expression levels in existing models of GCD2 disease, an ex vivo model of mutation-expressing primary corneal epithelial cells generated from corneal limbal biopsies from GCD2 patients would be more suitable than existing transgenic mouse models for future pre-clinical work in the development of gene silencing therapies for corneal dystrophies.

Original language	English
Article number	113681
Journal	Journal of Controlled Release
Volume	382
ISSN	0168-3659
DOIs	https://doi.org/10.1016/j.jconrel.2025.113681
Publication status	Published - Jun 2025

Keywords

Autosomal dominant
Granular corneal dystrophy type 2
R124H TGFBI
Silicon stabilized hybrid lipid nanoparticles
siRNA

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10.1016/j.jconrel.2025.113681

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McLain, A., Kowalczyk, A., Baran-Rachwalska, P., Sutera, F. M., Robertson, L. J., Nielsen, N. S., Enghild, J. J., Cobice, D., Bonelli, F., Barbaro, V., Ferrari, S., Patterson, B., Moore, L., Marshall, J., Nesbit, M. A., & Moore, T. (2025). TGFBI R124H mutant allele silencing in granular corneal dystrophy type 2 using topical siRNA delivery. Journal of Controlled Release, 382, Article 113681. https://doi.org/10.1016/j.jconrel.2025.113681

@article{8a59060451774f2abfbe087d559122bb,

title = "TGFBI R124H mutant allele silencing in granular corneal dystrophy type 2 using topical siRNA delivery",

abstract = "In recent years, success has been achieved in treating several eye conditions with oligonucleotide-based therapies. Herein, we outline the experimentation involved in progressing selection and development of a lead therapeutic siRNA for R124H mutation of TGFBI gene which causes Granular Corneal Dystrophy Type 2 (GCD2/Avellino CD). Firstly, a series of siRNA designs, generated by a gene walk across the R124H TGFBI mutation site, were tested and a lead siRNA identified. The lead siRNA was delivered into an immortalised human corneal epithelial cell line to assess on-target efficacy and off-target effects. The in vivo efficacy of the lead R124H TGFBI siRNA, complexed with Bio-Courier technology, silicon stabilized hybrid lipid nanoparticles (sshLNP), was assessed in a mouse model of GCD2 which expressed the human R124H TGFBI transgene. Following topical siRNA application for 5 consecutive days, expression of the R124H mutant TGFBI transgene was measured and shown to be reduced by 22.4 % (± 15.7 %, p < 0.05). We investigated gene expression in the mouse cornea and showed expression of murine Tgfbi was 20-fold lower than TGFBI in human cornea, and expression of the mutant TGFBI transgene was a further 3-fold lower. This estimated 60-fold lower mutant transgene expression may explain the low frequency of corneal deposits observed in this mouse model, limiting its usefulness to test whether siRNA silencing is capable of phenotypic improvement or regression of GCD2/Avellino corneal dystrophy. We assessed WT TGFBI silencing in human primary corneal epithelial cells (PCEC) derived from human corneal limbal biopsy material, which express TGFBI at a similar level to human corneal biopsy. We demonstrated that a single 100 nM siRNA treatment, delivered by the sshLNP to the primary human corneal epithelial cells, gave 26.6 % (± 6.6 %, p < 0.001) reduction in TGFBI mRNA and a 15.4 % (±10.5 %, p < 0.05 %) reduction in TGFBi protein after 48 h. In consideration of the mutant gene expression levels in existing models of GCD2 disease, an ex vivo model of mutation-expressing primary corneal epithelial cells generated from corneal limbal biopsies from GCD2 patients would be more suitable than existing transgenic mouse models for future pre-clinical work in the development of gene silencing therapies for corneal dystrophies.",

keywords = "Autosomal dominant, Granular corneal dystrophy type 2, R124H TGFBI, Silicon stabilized hybrid lipid nanoparticles, siRNA",

author = "Andrew McLain and Amanda Kowalczyk and Paulina Baran-Rachwalska and Sutera, {Flavia Maria} and Robertson, {Louise J.} and Nielsen, {Nadia Sukusu} and Enghild, {Jan J.} and Diego Cobice and Filippo Bonelli and Vanessa Barbaro and Stefano Ferrari and Benjamin Patterson and Luca Moore and John Marshall and Nesbit, {M. Andrew} and Tara Moore",

note = "Publisher Copyright: {\textcopyright} 2025",

year = "2025",

month = jun,

doi = "10.1016/j.jconrel.2025.113681",

language = "English",

volume = "382",

journal = "Journal of Controlled Release",

issn = "0168-3659",

publisher = "Elsevier B.V.",

}

McLain, A, Kowalczyk, A, Baran-Rachwalska, P, Sutera, FM, Robertson, LJ, Nielsen, NS , Enghild, JJ, Cobice, D, Bonelli, F, Barbaro, V, Ferrari, S, Patterson, B, Moore, L, Marshall, J, Nesbit, MA & Moore, T 2025, 'TGFBI R124H mutant allele silencing in granular corneal dystrophy type 2 using topical siRNA delivery', Journal of Controlled Release, vol. 382, 113681. https://doi.org/10.1016/j.jconrel.2025.113681

TGFBI R124H mutant allele silencing in granular corneal dystrophy type 2 using topical siRNA delivery. / McLain, Andrew; Kowalczyk, Amanda; Baran-Rachwalska, Paulina et al.
In: Journal of Controlled Release, Vol. 382, 113681, 06.2025.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - TGFBI R124H mutant allele silencing in granular corneal dystrophy type 2 using topical siRNA delivery

AU - McLain, Andrew

AU - Kowalczyk, Amanda

AU - Baran-Rachwalska, Paulina

AU - Sutera, Flavia Maria

AU - Robertson, Louise J.

AU - Nielsen, Nadia Sukusu

AU - Enghild, Jan J.

AU - Cobice, Diego

AU - Bonelli, Filippo

AU - Barbaro, Vanessa

AU - Ferrari, Stefano

AU - Patterson, Benjamin

AU - Moore, Luca

AU - Marshall, John

AU - Nesbit, M. Andrew

AU - Moore, Tara

PY - 2025/6

Y1 - 2025/6

N2 - In recent years, success has been achieved in treating several eye conditions with oligonucleotide-based therapies. Herein, we outline the experimentation involved in progressing selection and development of a lead therapeutic siRNA for R124H mutation of TGFBI gene which causes Granular Corneal Dystrophy Type 2 (GCD2/Avellino CD). Firstly, a series of siRNA designs, generated by a gene walk across the R124H TGFBI mutation site, were tested and a lead siRNA identified. The lead siRNA was delivered into an immortalised human corneal epithelial cell line to assess on-target efficacy and off-target effects. The in vivo efficacy of the lead R124H TGFBI siRNA, complexed with Bio-Courier technology, silicon stabilized hybrid lipid nanoparticles (sshLNP), was assessed in a mouse model of GCD2 which expressed the human R124H TGFBI transgene. Following topical siRNA application for 5 consecutive days, expression of the R124H mutant TGFBI transgene was measured and shown to be reduced by 22.4 % (± 15.7 %, p < 0.05). We investigated gene expression in the mouse cornea and showed expression of murine Tgfbi was 20-fold lower than TGFBI in human cornea, and expression of the mutant TGFBI transgene was a further 3-fold lower. This estimated 60-fold lower mutant transgene expression may explain the low frequency of corneal deposits observed in this mouse model, limiting its usefulness to test whether siRNA silencing is capable of phenotypic improvement or regression of GCD2/Avellino corneal dystrophy. We assessed WT TGFBI silencing in human primary corneal epithelial cells (PCEC) derived from human corneal limbal biopsy material, which express TGFBI at a similar level to human corneal biopsy. We demonstrated that a single 100 nM siRNA treatment, delivered by the sshLNP to the primary human corneal epithelial cells, gave 26.6 % (± 6.6 %, p < 0.001) reduction in TGFBI mRNA and a 15.4 % (±10.5 %, p < 0.05 %) reduction in TGFBi protein after 48 h. In consideration of the mutant gene expression levels in existing models of GCD2 disease, an ex vivo model of mutation-expressing primary corneal epithelial cells generated from corneal limbal biopsies from GCD2 patients would be more suitable than existing transgenic mouse models for future pre-clinical work in the development of gene silencing therapies for corneal dystrophies.

AB - In recent years, success has been achieved in treating several eye conditions with oligonucleotide-based therapies. Herein, we outline the experimentation involved in progressing selection and development of a lead therapeutic siRNA for R124H mutation of TGFBI gene which causes Granular Corneal Dystrophy Type 2 (GCD2/Avellino CD). Firstly, a series of siRNA designs, generated by a gene walk across the R124H TGFBI mutation site, were tested and a lead siRNA identified. The lead siRNA was delivered into an immortalised human corneal epithelial cell line to assess on-target efficacy and off-target effects. The in vivo efficacy of the lead R124H TGFBI siRNA, complexed with Bio-Courier technology, silicon stabilized hybrid lipid nanoparticles (sshLNP), was assessed in a mouse model of GCD2 which expressed the human R124H TGFBI transgene. Following topical siRNA application for 5 consecutive days, expression of the R124H mutant TGFBI transgene was measured and shown to be reduced by 22.4 % (± 15.7 %, p < 0.05). We investigated gene expression in the mouse cornea and showed expression of murine Tgfbi was 20-fold lower than TGFBI in human cornea, and expression of the mutant TGFBI transgene was a further 3-fold lower. This estimated 60-fold lower mutant transgene expression may explain the low frequency of corneal deposits observed in this mouse model, limiting its usefulness to test whether siRNA silencing is capable of phenotypic improvement or regression of GCD2/Avellino corneal dystrophy. We assessed WT TGFBI silencing in human primary corneal epithelial cells (PCEC) derived from human corneal limbal biopsy material, which express TGFBI at a similar level to human corneal biopsy. We demonstrated that a single 100 nM siRNA treatment, delivered by the sshLNP to the primary human corneal epithelial cells, gave 26.6 % (± 6.6 %, p < 0.001) reduction in TGFBI mRNA and a 15.4 % (±10.5 %, p < 0.05 %) reduction in TGFBi protein after 48 h. In consideration of the mutant gene expression levels in existing models of GCD2 disease, an ex vivo model of mutation-expressing primary corneal epithelial cells generated from corneal limbal biopsies from GCD2 patients would be more suitable than existing transgenic mouse models for future pre-clinical work in the development of gene silencing therapies for corneal dystrophies.

KW - Autosomal dominant

KW - Granular corneal dystrophy type 2

KW - R124H TGFBI

KW - Silicon stabilized hybrid lipid nanoparticles

KW - siRNA

UR - http://www.scopus.com/inward/record.url?scp=105002138346&partnerID=8YFLogxK

U2 - 10.1016/j.jconrel.2025.113681

DO - 10.1016/j.jconrel.2025.113681

M3 - Journal article

C2 - 40185334

AN - SCOPUS:105002138346

SN - 0168-3659

VL - 382

JO - Journal of Controlled Release

JF - Journal of Controlled Release

M1 - 113681

ER -

TGFBI R124H mutant allele silencing in granular corneal dystrophy type 2 using topical siRNA delivery

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